The Role of Histo-Blood Group Antigens and Microbiota in Human Norovirus Replication in Zebrafish Larvae

ABSTRACT Human norovirus (HuNoV) is the major agent for viral gastroenteritis, causing >700 million infections yearly. Fucose-containing carbohydrates named histo-blood group antigens (HBGAs) are known (co)receptors for HuNoV. Moreover, bacteria of the gut microbiota expressing HBGA-like structures have shown an enhancing effect on HuNoV replication in an in vitro model. Here, we studied the role of HBGAs and the host microbiota during HuNoV infection in zebrafish larvae. Using whole-mount immunohistochemistry, we visualized the fucose expression in the zebrafish gut for the HBGA Lewis X [LeX, α(1,3)-fucose] and core fucose [α(1,6)-fucose]. Costaining of HuNoV-infected larvae proved colocalization of LeX and to a lower extent core fucose with the viral capsid protein VP1, indicating the presence of fucose residues on infected cells. Upon blocking of fucose expression by a fluorinated fucose analogue, HuNoV replication was strongly reduced. Furthermore, by comparing HuNoV replication in conventional and germfree zebrafish larvae, we found that the natural zebrafish microbiome does not have an effect on HuNoV replication, contrary to earlier reports about the human gut microbiome. Interestingly, monoassociation with the HBGA-expressing Enterobacter cloacae resulted in a minor decrease in HuNoV replication, which was not triggered by a stronger innate immune response. Overall, we show here that fucose has an essential role for HuNoV infection in zebrafish larvae, as in the human host, but their natural gut microbiome does not affect viral replication. IMPORTANCE Despite causing over 700 million infections yearly, many gaps remain in the knowledge of human norovirus (HuNoV) biology due to an historical lack of efficient cultivation systems. Fucose-containing carbohydrate structures, named histo-blood group antigens, are known to be important (co)receptors for viral entry in humans, while the natural gut microbiota is suggested to enhance viral replication. This study shows a conserved mechanism of entry for HuNoV in the novel zebrafish infection model, highlighting the pivotal opportunity this model represents to study entry mechanisms and identify the cellular receptor of HuNoV. Our results shed light on the interaction of HuNoV with the zebrafish microbiota, contributing to the understanding of the interplay between gut microbiota and enteric viruses. The ease of generating germfree animals that can be colonized with human gut bacteria is an additional advantage of using zebrafish larvae in virology. This small animal model constitutes an innovative alternative to high-severity animal models.

Line 120 In which cases? The studies mentioned in ref 36 and 37? Line 116 no = not? Line 202-206 The authors use the word "suggested" twice but these studies (Science 2x) "showed/proved" that this was the case for the cells/animal models that they used, with many controls. So please reword. Line 241 remove "clear" Line 270 like it is written now it seems that the ELISA data shows that "HuNoV enters and replicates in HBGA-expressing cells......" But this should be something like "collectively the data presented here shows that HuNoV enters and replicates in HBGA-expressing cells......" Line 365 The authors compare their findings to those with the B-cell model, however the B-cells do not have glycans that are bound by HuNoV, and without bacteria/synthetic HBGA there is no replication at all. This is very different from the zebrafish model, where there is efficient HuNoV replication and the presence of glycans that are bound by HuNoV.
Reviewer #2 (Comments for the Author): The manuscript by A. Cuvry et al. successfully demonstrate the expression of alpha1-2 and alpha1-6 fucosilated carbohydrates in zebrafish, as well as they role in HuNoV infection in this animal model. It also illustrates how bacteria expressing HBGA do not enhance HuNoV replication in early larval stage. The experiments are properly designed and executed. The scientific quality of this work is high, and the results are sound for the community. I only have some minor comments: 1-I am very skeptical with the high variability shown in the binding assay in Supplementary Figure 1. Some reasons are: -I would like to drag the attention of the authors to the crystal structures available for this strain in complex with HBGAs (https://doi.org/10.1128/JVI.02968-14) and with milk oligosaccharides (10.1128/JVI.03223-15). A simple superimposition of available crystal structures will show that carbohydrates show very similar (in some cases identical) interactions with the P domains, which strongly suggests similar affinities. As an example, please compare PDBs 4X0C (Lewis X) vs 4OP7 (B-tris).
-In addition, dissociation constants KDs are available for a related strain GII.4 Saga 2006 (GenBank AB447457). Saga and Sydney share a 95% VP1 identity, with 100% conservation of amino acids located in the binding pocket. Comparison of crystal structures of Saga (4X06) and of Sydney (4OP7) in complex with B-tris show again identical interactions. NMR data available for Saga P dimers reveals very similar KDs for A-and B-tris.
-Also, Lea and LeX possess almost identical 3D structural principles, which are in turn responsible for their recognition by hNoV (10.1016/j.coviro.2018.04.007). Why should they exhibit such a large difference in Abs?
In summary, these results need to be taken with care. In my opinion, the differences in Abs observed between HBGAs are likely due to variable multivalent presentation, rather than real affinity. That is, it is highly probably that the short and rigid acetylphenylenediamine link used by IsoSep AB to connect the carbohydrates with the HSA is influencing the outcome of the experiment. The authors should at least discuss it in the Supplementary Figure 1.
2-Line 281: What about bile acids? They have been shown to interact in different ways with several hNoV strains. Does it make any sense in the biology of zebrafish larvae? 3-It is always helpful to add the GenBank accession number of the hNoV construct overexpressed, in this case VLPs. To be more precise, I would like to know if the VLPs used in this study and named as "GII.4 2012 Sydney" are in fact Hu/GII.4/Sydney/NSW0514/2012/AU, with GenBank accession number JX459908.1. Please add. 4-Please swap order of appearance in the text of figure 2B and Figure 2C. The order should be the one of appearance in the text. Also correct Fig. 2 if required. 5-Line 219: "harvested each day pi.." Is pi a typo?

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"showed/proved" that this was the case for the cells/animal models that they used, with many controls. So please reword.
We have rephrased the text according to the reviewer's request but kept the term 'suggested' when referring to the mechanism of action as the underlying mechanisms have not been fully elucidated yet.

Line 241: remove "clear"
We have modified the text according to the reviewer's request.
10. Line 270: like it is written now it seems that the ELISA data shows that "HuNoV enters and replicates in HBGA-expressing cells......" But this should be something like "collectively the data presented here shows that HuNoV enters and replicates in HBGA-expressing cells......" Sentence has been modified to clarify our conclusion according to the reviewer's request.

Line 365: The authors compare their findings to those with the B-cell model, however the Bcells do not have glycans that are bound by HuNoV, and without bacteria/synthetic HBGA there is no replication at all. This is very different from the zebrafish model, where there is efficient HuNoV replication and the presence of glycans that are bound by HuNoV.
The B-cell model does indeed show little to no replication after removal of bacteria from the sample which is different from our zebrafish model. We do however want to point out in the text that in the B cells, merely addition of (heat-killed) E.cloacae had a potent restorative effect on HuNoV replication. Based on these observations, we expected also an enhancing effect in our zebrafish model, even though there already is a robust replication in conventional and germ-free larvae.